Monoclonal antibodies against orthopoxviruses

ABSTRACT

The present invention relates to monoclonal antibodies that bind or neutralize Orthopoxviruses. The invention provides such antibodies, fragments of such antibodies retaining B5 or A33 binding ability, fully human antibodies retaining B5 or A33 binding ability, and pharmaceutical compositions including such antibodies. The invention further provides for isolated nucleic acids encoding the antibodies of the invention and host cells transformed therewith. Additionally, the invention provides for prophylactic, therapeutic, and diagnostic methods employing the antibodies and nucleic acids of the invention.

RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityto International Application No. PCT/US2006/048832 filed Dec. 22, 2006,which designated the United States and was published in English, andthis application is a continuation of and claims the benefit of priorityto International Application No. PCT/US2006/048833 filed Dec. 22, 2006,which designated the United States and was published in English; whereinboth of the aforementioned international applications claim the benefitof priority to U.S. Provisional Application No. 60/779,855, filed Mar.7, 2006, U.S. Provisional Application No. 60/763,786, filed Jan. 30,2006, and U.S. Provisional Application No. 60/753,437, filed Dec. 22,2005. All of the aforementioned applications are hereby expresslyincorporated by reference in their entireties.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledNIH307_(—)001C1_Sequence_Listing.TXT, created Jun. 19, 2008, which is 27Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the field of immunology andspecifically to monoclonal antibodies that bind or neutralizeOrthopoxviruses.

BACKGROUND OF THE INVENTION

Concerns that variola (smallpox) virus might be used as a biologicalweapon have led to the recommendation of widespread vaccination withvaccinia virus (VACV) (Henderson, D. A. 1999 Science 283:1279-1282).While vaccination is generally safe and effective for prevention ofsmallpox, it is well documented that various adverse reactions inindividuals have been caused by vaccination with existing licensedvaccines (Fulginiti, V. A. et al. 2003 Clin Infect Dis 37:251-271).Vaccinia immune globulin (VIG) prepared from vaccinated humans hashistorically been used to treat adverse reactions arising from VACVimmunization (Kempe, C. H. 1960 Pediatrics 26:176-189; Feery, B. J.(1976) Vox Sang 31:68-76; Hopkins, R. J. et al. 2004 Clin Infect Dis39:759-766; Hopkins, R. J. & Lane, J. M. 2004 Clin Infect Dis39:819-826) and to date, VIG is still the only recommended treatment(Hopkins, R. J. et al. 2004 Clin Infect Dis 39:759-766; Hopkins, R. J. &Lane, J. M. 2004 Clin Infect Dis 39:819-826). However, VIG lots may havedifferent potencies and carry the potential to transmit other viralagents.

VACV is the prototype virus in the genus Orthopoxvirus, which includesvariola virus, the causative agent of smallpox. There are two majorforms of infectious VACV: intracellular mature virus (MV) andextracellular enveloped virus (EV). The majority of the MV remainswithin the cell until lysis, but some are wrapped in additionalmembranes and exocytosed as EV. Most EV remains attached to the outsideof the plasma membrane and is responsible for direct cell-to-cellspread; however some are released into the medium and can causecomet-like satellite plaques (Blasco, R. & Moss, B. 1992 J Virol66:4170-4179; Blasco, R. et al. 1993 J Virol 67:3319-3325). The EV isimportant for virus dissemination in vivo as well as in cultured cells(Payne, L. G. 1980 J Gen Virol 50:89-100; Smith, G. L. &Vanderplasschen, A. 1998 Adv Exp Med Biol 440:395-414). Because an EV isessentially an MV enclosed by an additional membrane, the two forms ofVACV have different outer proteins and bind to cells differently(Vanderplasschen, A. et al. 1998 J Gen Virol 79:877-887), thoughultimately only the proteins of the MV membrane mediate membrane fusion(Moss, B. 2005 Virology 344:48-54). B5 is one of five known EV-specificproteins and is highly conserved among different strains of VACV as wellas in other orthopoxviruses (Engelstad, M. et al 1992 Virology188:801-810; Isaacs, S, N. et al. 1992 J Virol 66:7217-7224). B5 is a42-kDa glycosylated type I membrane protein with a large ectodomaincomposed of four small domains that are similar to short consensusrepeat (SCR) domains of complement regulatory protein (Engelstad, M. etal 1992 Virology 188:801-810; Isaacs, S, N. et al. 1992 J Virol66:7217-7224) although no complement regulatory activity has beendemonstrated. B5 is required for efficient envelopment of MV, as well asfor actin tail formation, normal plaque size and virulence (Engelstad,M. & Smith, G. L. 1993 Virology 194:627-637; Sanderson, C. M. et al.1998 J Gen Virol 79:1415-1425; Wolffe, E. J. et al. 1993 J Virol67:4732-4741).

The B5 protein is an important target for neutralizing antibodies:antisera to B5 can neutralize EV in a plaque reduction assay and inhibit“comet formation”, the in vitro manifestation of cell-to-cell spread ofEV (Engelstad, M. et al 1992 Virology 188:801-810, Galmiche, M. C. etal. 1999 Virology 254:71-80; Law, M. & Smith, G. L. 2001 Virology280:132-142; Aldaz-Carroll et al. 2005 J Virol 79:6260-6271). Recentstudies showed that anti-B5 in VIG was responsible for most of theneutralizing activity against EV as measured by a plaque reduction assay(Bell, E. et al. 2004 Virology 325:425-431). To date, rat and mouseanti-B5 neutralizing MAbs have been reported (Aldaz-Carroll et al. 2005J Virol 79:6260-6271; Schmelz, M. et al. 1994 J Virol 68:130-147) andthe epitopes recognized by mouse MAbs have been mapped to the border ofSCR1-SCR2 and/or the stalk of B5 (Aldaz-Carroll et al. 2005 J Virol79:6260-6271). In addition, a rat monoclonal antibody (MAb) to B5provided protection in a VACV mouse challenge model (Lustig, S. et al.2005 J Virol 79:13454-13462).

Segue to the Invention

We decided to obtain therapeutically useful high-affinity monoclonalantibodies to B5 protein from chimpanzees because of the extremesimilarity of their IgG with human IgG (Ehrlich, P. H. et al. 1990 HumAntibodies Hybridomas 1:23-26; Schofield, D. J. et al. 2002 Virology292:127-136). A phage display library bearing Fabs was derived from thebone marrow of chimpanzees that had been vaccinated with VACV. From thislibrary, we isolated and characterized two potent anti-B5 antibodiesthat neutralize variola virus in addition to VACV. Such human-likemonoclonal antibodies against B5 are contemplated as providing superiorprotection with a lower dose and higher safety profile than VIG.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies that bind orneutralize Orthopoxviruses. The invention provides such antibodies,fragments of such antibodies retaining B5 or A33 binding ability, fullyhuman antibodies retaining B5 or A33 binding ability, and pharmaceuticalcompositions including such antibodies. The invention further providesfor isolated nucleic acids encoding the antibodies of the invention andhost cells transformed therewith. Additionally, the invention providesfor prophylactic, therapeutic, and diagnostic methods employing theantibodies and nucleic acids of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequences of variable domains of heavy (8AH8AL—SEQ IDNO: 1 and 8AH7AL—SEQ ID NO: 17) (shown in FIG. 1A) and light (8AH8AL—SEQID NO: 9 and 8AH7AL—SEQ ID NO: 25) (shown in FIG. 1B) chains ofchimpanzee/human anti-B5 MAbs and ELISA titration of anti-B5 8AH8AL.Complementarity-determining regions (CDR1, CDR2, and CDR3) and frameworkregions (FWR1, FWR2, FWR3, and FWR4) are indicated above the sequence orsequence alignment. Dashes indicate an identical residue. As shown inFIG. 1C, for the ELISA binding assay, the wells of ELISA plates werecoated with recombinant B5 (275t) or unrelated proteins (BSA,thyroglobulin, lysozyme, and phosphorylase-b) and then incubated with8AH8AL at various concentrations. Bound IgG was detected by the additionof peroxidase-conjugated anti-human (Fab)₂ followed by TMB substrate.

FIG. 2. Epitope mapping by Western blotting. Similar amounts ofdifferent-sized fragments of B5 expressed in bacteria were blotted ontothe membrane and anti-B5 MAb was added as shown in FIG. 2A. The boundanti-B5 was detected by HRP-conjugated anti-human IgG (Fab′)₂. Thepositive bands were visualized with addition of LumiGLO chemiluminescentperoxidase substrate and exposing the membrane to X-ray film. The resultwas summarized in FIG. 2B, where the peptides that reacted with antibodywere scored as positive (+). Faint intensity of the bands was scored as+/−. The numbers denote the starting and ending amino acid.

FIG. 3. In vitro neutralizing activity of anti-B5 MAbs, measured by acomet-reduction assay. In FIG. 3A, BS-C-1 cells were infected withapproximately 50 plaque-forming units of VACV, strain IHD-J. After 2 hat 37° C., the monolayer was washed and fresh medium containingindicated amounts of chimpanzee anti-B5 8AH7AL or 8AH8AL was added. PBSand rabbit hyper-immune serum served as negative and positive controls,respectively. After 48 h, the monolayers were stained with crystalviolet. For the smallpox assay of FIG. 3B, monolayers of BS-C-40 cellsin 6-well cell culture plates were infected with the Solaimen strain ofvariola virus at 50 plaque-forming units per well in RPMI mediumcontaining 2% FBS. After 1 h, the medium was aspirated; cells werewashed twice, and overlaid with RPMI containing 25 μg, 2.5 μg, or 0 μgof anti-B5 IgG. The plates were then incubated in a CO₂ incubator for 4days at 35.5° C. Cells were fixed and reacted with polyclonal rabbitanti-variola virus antibody. Following incubation with goatanti-rabbit-HRP conjugate, comets were visualized by addition of TruBlueperoxidase substrate.

FIG. 4. Prophylactic and therapeutic protection in mice by anti-B5 MAbs.Groups of five BALB/c mice were inoculated intraperitoneally with 90 μgof purified IgG (FIG. 4A), or different amounts of IgG (FIG. 4B). Twentyfour hours later, mice were challenged intranasally with 10⁵plaque-forming units (pfu) of the WR strain of vaccinia virus. Ninetymicrograms of rat anti-B5 19C2 IgG (FIG. 4A) or 5 mg of human vacciniaimmune globulin (VIG) (FIG. 4B) were used for comparison. In FIG. 4C,groups of five BALB/c mice were inoculated intranasally with 10⁵ pfu ofvaccinia virus, strain WR. After 48 h, the mice were injectedintraperitoneally with 90 μg of purified IgG, or 5 mg of human VIG. Micewere weighed individually and mean percentages of startingweight±standard error were plotted. Controls were unimmunized (noantibody) or unchallenged (no virus). †, died naturally or killedbecause of 30% weight loss.

FIG. 5. Antibody responses elicited by challenge with the WR strain ofVACV. Mice were bled 22 days after challenge with WR. Individual serawere assayed for binding to EV-associated proteins: B5 (FIG. 5A), andA33 (FIG. 5B); and two MV-associated proteins: L1 (FIG. 5C) and A27(FIG. 5D). The sera were also assayed for neutralizing antibodies to MV(FIG. 5E). IC₅₀: the reciprocal serum dilution that can neutralize 50%of virus. Reciprocal endpoint binding titers were determined by ELISAusing anti-mouse peroxidase. Filled and open bars represent animalsimmunized with 8AH8AL and VIG, respectively. Those groups that receivedpost-exposure immunization are indicated by “-post”.

FIG. 6. Sequences of (FIG. 6A) VH (6C—SEQ ID NO: 33; 12C—SEQ ID NO: 49;and 12F—SEQ ID NO: 65) and (FIG. 6B) (6C—SEQ ID NO: 41; 12C—SEQ ID NO:57; and 12F—SEQ ID NO: 73) VL of MAbs against A33 proteins of vacciniavirus.

FIG. 7. Binding specificity of anti-A33 MAb 6C.

FIG. 8. Epitope mapping of anti-A33 MAb.

FIG. 9. Comet reduction assay for (FIG. 9A) vaccinia virus with anti-A33MAbs and (FIG. 9B) variola virus with anti-A33 MAb 6C.

FIG. 10. In vivo neutralization of extracellular mature vaccinia virusby chimpanzee/human monoclonal anti-A33: Challenged with 10⁵ virulentvaccinia viruses.

FIG. 11. Passive immunization of mice with chimpanzee/human monoclonalantibody 6C. Groups of seven-week old female BALB/c mice (TaconicBiotechnology, Germantown, N.Y.) were immunized with different amountsof monoclonal antibody 6C (90, 45, or 22.5 μg) or with 5 mg of vacciniaimmune globulin (VIG) (Cangene). Antibodies were diluted in PBS andinjected by the intraperitoneal route. Twenty four hours later, theanimals were challenged intranasally with 10⁵ PFU of vaccinia virus WR.Mice were weighed daily for 14 days and were sacrificed if their weightdiminished to 70% of the initial weight, in accordance with NIAID AnimalCare and Use protocols.

FIG. 12. Post-exposure treatment of mice with chimpanzee/humanmonoclonal antibody 6C. Groups of seven-week old female BALB/c mice(Taconic Biotechnology, Germantown, N.Y.) were challenged intranasallywith 10⁵ PFU of vaccinia virus WR. Forty eight hours later, either 6C(90 μg) or VIG (5 mg) was administered by the intraperitoneal route.Mice were weighed daily for 14 days and were sacrificed if their weightdiminished to 70% of the initial weight, in accordance with NIAID AnimalCare and Use protocols.

FIG. 13. Passive immunization of mice with chimpanzee/human monoclonalantibodies 6C and/or 8AH8AL. Groups of seven-week old female BALB/c mice(Taconic Biotechnology, Germantown, N.Y.) were immunized by theintraperitoneal route with 90 μg of 6C, 90 μg of 8AH8AL, 45 μg each ofthese antibodies, or 5 mg of VIG. Twenty four hours later, mice werechallenged intranasally with 5×10⁵ PFU of vaccinia virus WR. Mice wereweighed daily for 14 days and were sacrificed if their weight diminishedto 70% of the initial weight, in accordance with NIAID Animal Care andUse protocols.

FIG. 14. Post-exposure treatment of mice with chimpanzee/humanmonoclonal antibodies 6C and/or 8AH8AL. Groups of seven-week old femaleBALB/c mice (Taconic Biotechnology, Germantown, N.Y.) were challengedintranasally with 5×10⁵ PFU of vaccinia virus WR. Forty eight hourslater either 90 μg of 6C, 90 μg of 8AH8AL, 45 μg each of theseantibodies, or 5 mg of VIG was administered by the intraperitonealroute. Mice were weighed daily for 14 days and were sacrificed if theirweight diminished to 70% of the initial weight, in accordance with NIAIDAnimal Care and Use protocols.

FIG. 15. Passive immunization of mice with chimpanzee/human monoclonalantibodies 6C or 12F. Groups of seven-week old female BALB/c mice(Taconic Biotechnology, Germantown, N.Y.) were immunized by theintraperitoneal route with either 90 μg (FIG. 15A), 45 μg (FIG. 15B), or22.5 μg (FIG. 15C) of antibody 6C or 12F, or with 5 mg of VIG. Twentyfour hours later, mice were challenged intranasally with 5×10⁴ PFU ofvaccinia virus WR. Mice were weighed daily for 16 days and weresacrificed if their weight diminished to 70% of the initial weight, inaccordance with NIAID Animal Care and Use protocols.

TABLE A Brief Description of Mab SEQ ID NOs Heavy Chain SEQ ID NOs LightChain SEQ ID NOs Fab/mab V_(H) FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 V_(L) FR1CDR1 FR2 CDR2 FR3 CDR3 FR4 Anti-B5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1516 8AH8AL Anti-B5 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 8AH7ALAnti-A33 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 6C Anti-A33 4950 51 52 53 54 55 56 57 58 59 60 61 62 63 64 12C Anti-A33 65 66 67 68 6970 71 72 73 74 75 76 77 78 79 80 12F

Deposit of Biological Material

The following biological material has been deposited in accordance withthe terms of the Budapest Treaty with the American Type CultureCollection (ATCC), Manassas, Va., on the date indicated:

Biological material Designation No. Date Chimpanzee anti-vaccinia virusB5 PTA-7294 Dec. 22, 2005 protein Fab in pComb 3H vector, 8AH8AL

Chimpanzee anti-vaccinia virus B5 protein Fab in pComb 3H vector,8AH8AL, was deposited as ATCC Accession No. PTA-7294 on Dec. 22, 2005with the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA. This deposit was made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure andthe Regulations there under (Budapest Treaty). This assures maintenanceof a viable culture of the deposit for 30 years from date of deposit.The deposit will be made available by ATCC under the terms of theBudapest Treaty, and subject to an agreement between Applicant and ATCCwhich assures permanent and unrestricted availability of the progeny ofthe culture of the deposit to the public upon issuance of the pertinentU.S. patent or upon laying open to the public of any U.S. or foreignpatent application, whichever comes first, and assures availability ofthe progeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.14).Availability of the deposited biological material is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

Biological material Designation No. Date Chimpanzee anti-vaccinia virusA33 PTA-7323 Jan. 18, 2006 protein Fab in pComb 3H vector, 6C

Chimpanzee anti-vaccinia virus A33 protein Fab in pComb 3H vector, 6C,was deposited as ATCC Accession No. PTA-7323 on Jan. 18, 2006 with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, USA. This deposit was made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure andthe Regulations there under (Budapest Treaty). This assures maintenanceof a viable culture of the deposit for 30 years from date of deposit.The deposit will be made available by ATCC under the terms of theBudapest Treaty, and subject to an agreement between Applicant and ATCCwhich assures permanent and unrestricted availability of the progeny ofthe culture of the deposit to the public upon issuance of the pertinentU.S. patent or upon laying open to the public of any U.S. or foreignpatent application, whichever comes first, and assures availability ofthe progeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.14).Availability of the deposited biological material is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

Biological material Designation No. Date Chimpanzee anti-vaccinia virusA33 PTA-7324 Jan. 18, 2006 protein Fab in pComb 3H vector, 12F

Chimpanzee anti-vaccinia virus A33 protein Fab in pComb 3H vector, 12F,was deposited as ATCC Accession No. PTA-7324 on Jan. 18, 2006 with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209, USA. This deposit was made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure andthe Regulations there under (Budapest Treaty). This assures maintenanceof a viable culture of the deposit for 30 years from date of deposit.The deposit will be made available by ATCC under the terms of theBudapest Treaty, and subject to an agreement between Applicant and ATCCwhich assures permanent and unrestricted availability of the progeny ofthe culture of the deposit to the public upon issuance of the pertinentU.S. patent or upon laying open to the public of any U.S. or foreignpatent application, whichever comes first, and assures availability ofthe progeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC §122 and theCommissioner's rules pursuant thereto (including 37 CFR §1.14).Availability of the deposited biological material is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Vaccinia Virus StrainWR

The complete genome sequence of the vaccinia virus strain WR, includingB5R (B5) and A33R (A33), can be obtained at Genbank Accession No.AY243312.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g., Singleton P andSainsbury D., Dictionary of Microbiology and Molecular Biology 3^(rd)ed., J. Wiley & Sons, Chichester, New York, 2001, and Fields Virology4^(th) ed., Knipe D. M. and Howley P. M. eds, Lippincott Williams &Wilkins, Philadelphia 2001.

As used herein, the term “antibody” means an immunoglobulin molecule ora fragment of an immunoglobulin molecule having the ability tospecifically bind to a particular antigen. Antibodies are well known tothose of ordinary skill in the science of immunology. As used herein,the term “antibody” means not only full-length antibody molecules butalso fragments of antibody molecules retaining antigen binding ability.Such fragments are also well known in the art and are regularly employedboth in vitro and in vivo. In particular, as used herein, the term“antibody” means not only full-length immunoglobulin molecules but alsoantigen binding active fragments such as the well-known active fragmentsF(ab′)₂, Fab, Fv, and Fd.

As used herein, the term “Orthopoxvirus infection” means an infection byan Orthopoxvirus. Four Orthopoxvirus species; three of which arezoonotic, may infect humans: (a) variola virus, the cause of smallpox,which is eradicated, is a strictly human pathogen producing a febrilepustular rash illness; (b) monkeypox virus, which is reported from nineWest and Central African rainforest countries, mainly the DemocraticRepublic of Congo (DRC, formerly Zaire), is a zoonotic agent of asmallpox-like illness of low interhuman transmissibility; (c) cowpoxvirus is rodent-borne and indigenous to rodents in several European anda few western Asian countries, where humans appear to acquire alocalized pustular skin infection by contact with infected rodents, ormore likely, intermediate hosts, such as pet cats, cows, or otheranimals; and (d) the vaccinia virus subspecies buffalopox virus, whichoccurs mainly on the Indian subcontinent, causes localized oral and skinlesions after contact with infected dairy animals or drinking theirmilk. In addition, vaccinia virus strains used for smallpox vaccinationform a dermal pustule and may cause postvaccinal side effects.

As used herein with respect to polypeptides, the term “substantiallypure” means that the polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use. Inparticular, the polypeptides are sufficiently pure and are sufficientlyfree from other biological constituents of their host's cells so as tobe useful in, for example, generating antibodies, sequencing, orproducing pharmaceutical preparations. By techniques well known in theart, substantially pure polypeptides may be produced in light of thenucleic acid and amino acid sequences disclosed herein. Because asubstantially purified polypeptide of the invention may be admixed witha pharmaceutically acceptable carrier in a pharmaceutical preparation,the polypeptide may comprise only a certain percentage by weight of thepreparation. The polypeptide is nonetheless substantially pure in thatit has been substantially separated from the substances with which itmay be associated in living systems.

As used herein with respect to nucleic acids, the term “isolated” means:(1) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences, as desired.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmidsand phagemids. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification and selection of cells which have beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

Novel Anti-B5 and Anti-A33 Monoclonal Antibodies

The present invention derives, in part, from the isolation andcharacterization of novel chimpanzee Fab fragments and their humanizedmonoclonal antibodies that selectively bind Orthopoxvirus B5 or A33antigens. Additionally, these new monoclonal antibodies have been shownto neutralize Orthopoxviruses. The paratopes of the anti-B5 and anti-A33Fab fragments associated with the neutralization epitope on the B5 andA33 antigens are defined by the amino acid (aa) sequences of theimmunoglobulin heavy and light chain V-regions described in FIGS. 1 and6 and in SEQ ID NO: 1 through SEQ ID NO: 80 of Table A. The nucleic acidsequences coding for these amino acid sequences were identified bysequencing the Fab heavy chain and light chain fragments. Due to thedegeneracy of the DNA code, the paratope is more properly defined by thederived amino acid sequences depicted in FIGS. 1 and 6 and in SEQ ID NO:1 through SEQ ID NO: 80 of Table A.

In one set of embodiments, the present invention provides thefull-length, humanized anti-B5 or anti-A33 monoclonal antibodies inisolated form and in pharmaceutical preparations. Similarly, asdescribed herein, the present invention provides isolated nucleic acids,host cells transformed with nucleic acids, and pharmaceuticalpreparations including isolated nucleic acids, encoding the full-length,humanized monoclonal antibody of the anti-B5 or anti-A33 monoclonalantibodies. Finally, the present invention provides methods, asdescribed more fully herein, employing these antibodies and nucleicacids in the in vitro and in vivo diagnosis, prevention and therapy ofOrthopoxvirus infection.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of afull-length antibody. Similarly, an antibody from which the Fc regionhas been enzymatically cleaved, or which has been produced without theFc region, designated an Fab fragment, retains one of the antigenbinding sites of a full-length antibody molecule. Proceeding further,Fab fragments consist of a covalently bound antibody light chain and aportion of the antibody heavy chain denoted Fd. The Fd fragments are themajor determinant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986, supra; Roitt, 1991, supra). In both the heavychain Fd fragment and the light chain of IgG immunoglobulins, there arefour framework regions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

The complete amino acid sequences of the antigen-binding Fab portion ofthe anti-B5 or anti-A33 monoclonal antibodies as well as the relevant FRand CDR regions are disclosed herein. SEQ. ID. NOs: 1, 17, 33, 49, and65 disclose the amino acid sequence of the Fd fragment of anti-B5 oranti-A33 monoclonal antibodies. The amino acid sequences of the heavychain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions are disclosed as(FR1, SEQ. ID. NO: 2, 18, 34, 50, and 66); (CDR1, SEQ ID NOs: 3, 19, 35,51, and 67); (FR2, SEQ ID NOs: 4, 20, 36, 52 and 68); (CDR2, SEQ ID NOs:5, 21, 37, 53 and 69); (FR3, SEQ ID NOs: 6, 22, 38, 54 and 70); (CDR3,SEQ ID NOs: 7, 23, 39, 55 and 71); and (FR4, SEQ ID NOs: 8, 24, 40, 56and 72). SEQ ID NOs: 9, 25, 41, 57 and 73 disclose the amino acidsequences of the light chains of the anti-B5 or anti-A33 monoclonalantibodies. The amino acid sequences of the light chain FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4 regions are disclosed as (FR1, SEQ ID NOs: 10,26, 42, 58 and 74); (CDR1, SEQ ID NOs: 11, 27, 43, 59 and 75); (FR2, SEQID NOs: 12, 28, 44, 60 and 76); (CDR2, SEQ ID NOs: 13, 29, 45, 61 and77); (FR3, SEQ ID NOs: 14, 30, 46, 62 and 78); (CDR3, SEQ ID NOs: 15,31, 47, 63 and 79); (FR4, SEQ ID NOs: 16, 32, 48, 64 and 80).

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of full-length antibodies with antigen-binding ability, areoften referred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments ofthe 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 oranti-A33 antibody; chimeric antibodies in which the Fc and/or FR and/orCDR1 and/or CDR2 and/or light chain CDR3 regions of the 8AH8AL, 8AH7AL,6C, 12C, or 12F antibody, or other anti-B5 or anti-A33 antibody havebeen replaced by homologous human or non-human sequences; chimericF(ab′)₂ fragment antibodies in which the FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions of the 8AH8AL, 8AH7AL, 6C, 12C, or 12Fantibody, or other anti-B5 or anti-A33 antibody have been replaced byhomologous human or non-human sequences; chimeric Fab fragmentantibodies in which the FR and/or CDR1 and/or CDR2 and/or light chainCDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. Thus, those skilled in the art may alter the8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33antibody by the construction of CDR grafted or chimeric antibodies orantibody fragments containing all, or part thereof, of the disclosedheavy and light chain V-region CDR amino acid sequences (Jones, P. T. etal. 1986 Nature 321:522-525; Verhoeyen, M. et al. 1988 Science39:1534-1536; and Tempest, P. R. et al. 1991 Bio/Technology 9:266-271),without destroying the specificity of the antibodies for the B5 or A33epitopes. Such CDR grafted or chimeric antibodies or antibody fragmentscan be effective in prevention and treatment of Orthopoxvirus infectionin animals (e.g. cattle) and man.

In preferred embodiments, the chimeric antibodies of the invention arefully human or humanized chimpanzee monoclonal antibodies including atleast the heavy chain CDR3 region of the 8AH8AL, 8AH7AL, 6C, 12C, or 12Fantibody, or other anti-B5 or anti-A33 antibody. As noted above, suchchimeric antibodies may be produced in which some or all of the FRregions of the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or otheranti-B5 or anti-A33 antibody have been replaced by other homologoushuman FR regions. In addition, the Fc portions may be replaced so as toproduce IgA or IgM as well as IgG antibodies bearing some or all of theCDRs of the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5or anti-A33 antibody. Of particular importance is the inclusion of theheavy chain CDR3 region and, to a lesser extent, the other CDRs of the8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33antibody. Such fully human or humanized chimpanzee monoclonal antibodieswill have particular utility in that they will not evoke an immuneresponse against the antibody itself.

It is also possible, in accordance with the present invention, toproduce chimeric antibodies including non-human sequences. Thus, one mayuse, for example, murine, ovine, equine, bovine or other mammalian Fc orFR sequences to replace some or all of the Fc or FR regions of the8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33antibody. Some of the CDRs may be replaced as well. Again, however, itis preferred that at least the heavy chain CDR3 of the 8AH8AL, 8AH7AL,6C, 12C, or 12F antibody, or other anti-B5 or anti-A33 antibody beincluded in such chimeric antibodies and, to a lesser extent, it is alsopreferred that some or all of the other CDRs of the 8AH8AL, 8AH7AL, 6C,12C, or 12F antibody, or other anti-B5 or anti-A33 antibody be included.Such chimeric antibodies bearing non-human immunoglobulin sequencesadmixed with the CDRs of the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody,or other anti-B5 or anti-A33 antibody are not preferred for use inhumans and are particularly not preferred for extended use because theymay evoke an immune response against the non-human sequences. They may,of course, be used for brief periods or in immunosuppressed individualsbut, again, fully human or humanized chimpanzee monoclonal antibodiesare preferred. Because such antibodies may be used for brief periods orin immunosuppressed subjects, chimeric antibodies bearing non-humanmammalian Fc and FR sequences but including at least the heavy chainCDR3 of the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5or anti-A33 antibody are contemplated as alternative embodiments of thepresent invention.

For inoculation or prophylactic uses, the antibodies of the presentinvention are preferably full-length antibody molecules including the Fcregion. Such full-length antibodies will have longer half-lives thansmaller fragment antibodies (e.g. Fab) and are more suitable forintravenous, intraperitoneal, intramuscular, intracavity, subcutaneous,or transdermal administration.

In some embodiments, Fab fragments, including chimeric Fab fragments,are preferred. Fabs offer several advantages over F(ab′)₂ and wholeimmunoglobulin molecules for this therapeutic modality. First, becauseFabs have only one binding site for their cognate antigen, the formationof immune complexes is precluded whereas such complexes can be generatedwhen bivalent F(ab′)₂ s and whole immunoglobulin molecules encountertheir target antigen. This is of some importance because immune complexdeposition in tissues can produce adverse inflammatory reactions.Second, because Fabs lack an Fc region they cannot trigger adverseinflammatory reactions that are activated by Fc, such as activation ofthe complement cascade. Third, the tissue penetration of the small Fabmolecule is likely to be much better than that of the larger wholeantibody. Fourth, Fabs can be produced easily and inexpensively inbacteria, such as E. coli, whereas whole immunoglobulin antibodymolecules require mammalian cells for their production in usefulamounts. The latter entails transfection of immunoglobulin sequencesinto mammalian cells with resultant transformation. Amplification ofthese sequences must then be achieved by rigorous selective proceduresand stable transformants must be identified and maintained. The wholeimmunoglobulin molecules must be produced by stably transformed, highexpression mammalian cells in culture with the attendant problems ofserum-containing culture medium. In contrast, production of Fabs in E.coli eliminates these difficulties and makes it possible to producethese antibody fragments in large fermenters which are less expensivethan cell culture-derived products.

In addition to Fabs, smaller antibody fragments and epitope-bindingpeptides having binding specificity for the epitope defined by the8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33antibody are also contemplated by the present invention and can also beused to bind or neutralize the virus. For example, single chainantibodies can be constructed according to the method of U.S. Pat. No.4,946,778, to Ladner et al. Single chain antibodies comprise thevariable regions of the light and heavy chains joined by a flexiblelinker moiety. Yet smaller is the antibody fragment known as the singledomain antibody or Fd, which comprises an isolated VH single domain.Techniques for obtaining a single domain antibody with at least some ofthe binding specificity of the full-length antibody from which they arederived are known in the art.

It is possible to determine, without undue experimentation, if analtered or chimeric antibody has the same specificity as the 8AH8AL,8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33 antibodyof the invention by ascertaining whether the former blocks the latterfrom binding to B5 or A33 antigen. If the monoclonal antibody beingtested competes with the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, orother anti-B5 or anti-A33 antibody, as shown by a decrease in binding ofthe 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 oranti-A33 antibody, then it is likely that the two monoclonal antibodiesbind to the same, or a closely spaced, epitope. Still another way todetermine whether a monoclonal antibody has the specificity of the8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 or anti-A33antibody of the invention is to pre-incubate the 8AH8AL, 8AH7AL, 6C,12C, or 12F antibody, or other anti-B5 or anti-A33 antibody with B5 orA33 antigen with which it is normally reactive, and then add themonoclonal antibody being tested to determine if the monoclonal antibodybeing tested is inhibited in its ability to bind B5 or A33 antigen. Ifthe monoclonal antibody being tested is inhibited then, in alllikelihood, it has the same, or a functionally equivalent, epitope andspecificity as the 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or otheranti-B5 or anti-A33 antibody of the invention. Screening of monoclonalantibodies of the invention also can be carried out utilizing anOrthopoxvirus and determining whether the monoclonal antibodyneutralizes the virus.

By using the antibodies of the invention, it is now possible to produceanti-idiotypic antibodies which can be used to screen other monoclonalantibodies to identify whether the antibody has the same bindingspecificity as an antibody of the invention. In addition, suchantiidiotypic antibodies can be used for active immunization (Herlyn, D.et al. 1986 Science 232:100-102). Such anti-idiotypic antibodies can beproduced using well-known hybridoma techniques (Kohler, G. and Milstein,C. 1975 Nature 256:495-497). An anti-idiotypic antibody is an antibodywhich recognizes unique determinants present on the monoclonal antibodyproduced by the cell line of interest. These determinants are located inthe hypervariable region of the antibody. It is this region which bindsto a given epitope and, thus, is responsible for the specificity of theantibody.

An anti-idiotypic antibody can be prepared by immunizing an animal withthe monoclonal antibody of interest. The immunized animal will recognizeand respond to the idiotypic determinants of the immunizing antibody andproduce an antibody to these idiotypic determinants. By using theanti-idiotypic antibodies of the immunized animal, which are specificfor the monoclonal antibodies of the invention, it is possible toidentify other clones with the same idiotype as the antibody of thehybridoma used for immunization. Idiotypic identity between monoclonalantibodies of two cell lines demonstrates that the two monoclonalantibodies are the same with respect to their recognition of the sameepitopic determinant. Thus, by using anti-idiotypic antibodies, it ispossible to identify other hybridomas expressing monoclonal antibodieshaving the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is theimage of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

Nucleic Acids Encoding Anti-B5 or Anti-A33 Monoclonal Antibodies

Given the disclosure herein of the amino acid sequences of the heavychain Fd and light chain variable domains of the 8AH8AL, 8AH7AL, 6C,12C, or 12F antibody, or other anti-B5 or anti-A33 antibody, one ofordinary skill in the art is now enabled to produce nucleic acids whichencode these antibodies or which encode the various fragment antibodiesor chimeric antibodies described above. It is contemplated that suchnucleic acids will be operably joined to other nucleic acids forming arecombinant vector for cloning or for expression of the antibodies ofthe invention. The present invention includes any recombinant vectorcontaining the coding sequences, or part thereof, whether forprokaryotic or eukaryotic transformation, transfection or gene therapy.Such vectors may be prepared using conventional molecular biologytechniques, known to those with skill in the art, and would comprise DNAcoding sequences for the immunoglobulin V-regions of the 8AH8AL, 8AH7AL,6C, 12C, or 12F antibody, or other anti-B5 or anti-A33 antibody,including framework and CDRs or parts thereof, and a suitable promotereither with (Whittle, N. et al. 1987 Protein Eng. 1:499-505 and Burton,D. R. et al. 1994 Science 266:1024-1027) or without (Marasco, W. A. etal. 1993 Proc. Natl. Acad, Sci. (USA) 90:7889-7893 and Duan, L. et al.1994 Proc. Natl. Acad, Sci. (USA) 91:5075-5079) a signal sequence forexport or secretion. Such vectors may be transformed or transfected intoprokaryotic (Huse, W. D. et al. 1989 Science 246:1275-1281; Ward, S. etal. 1989 Nature 341:544-546; Marks, J. D. et al. 1991 J. Mol. Biol.222:581-597; and Barbas, C. F. et al. 1991 Proc. Natl. Acad. Sci. (USA)88:7978-7982) or eukaryotic (Whittle, N. et al. 1987 Protein Eng.1:499-505 and Burton, D. R. et al. 1994 Science 266:1024-1027) cells orused for gene therapy (Marasco, W. A. et al. 1993 Proc. Natl. Acad, Sci.(USA) 90:7889-7893 and Duan, L. et al. 1994 Proc. Natl. Acad, Sci. (USA)91:5075-5079) by conventional techniques, known to those with skill inthe art.

The expression vectors of the present invention include regulatorysequences operably joined to a nucleotide sequence encoding one of theantibodies of the invention. As used herein, the term “regulatorysequences” means nucleotide sequences which are necessary for orconducive to the transcription of a nucleotide sequence which encodes adesired polypeptide and/or which are necessary for or conducive to thetranslation of the resulting transcript into the desired polypeptide.Regulatory sequences include, but are not limited to, 5′ sequences suchas operators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals. The vectors of the invention mayoptionally include 5′ leader or signal sequences, 5′ or 3′ sequencesencoding fusion products to aid in protein purification, and variousmarkers which aid in the identification or selection of transformants.The choice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art. The subsequentpurification of the antibodies may be accomplished by any of a varietyof standard means known in the art.

A preferred vector for screening monoclonal antibodies, but notnecessarily preferred for the mass production of the antibodies of theinvention, is a recombinant DNA molecule containing a nucleotidesequence that codes for and is capable of expressing a fusionpolypeptide containing, in the direction of amino- to carboxy-terminus,(1) a prokaryotic secretion signal domain, (2) a polypeptide of theinvention, and, optionally, (3) a fusion protein domain. The vectorincludes DNA regulatory sequences for expressing the fusion polypeptide,preferably prokaryotic, regulatory sequences. Such vectors can beconstructed by those with skill in the art and have been described bySmith, G. P. et al. (1985 Science 228:1315-1317); Clackson, T. et al.(1991 Nature 352:624-628); Kang et al. (1991 in “Methods: A Companion toMethods in Enzymology: Vol. 2”; R. A. Lerner and D. R. Burton, ed.Academic Press, NY, pp 111-118); Barbas, C. F. et al. (1991 Proc, Natl.Acad. Sci, (USA) 88:7978-7982), Roberts, B. L. et al. (1992 Proc. Natl.Acad. Sci. (USA) 89:2429-2433).

A fusion polypeptide may be useful for purification of the antibodies ofthe invention. The fusion domain may, for example, include a poly-Histail which allows for purification on Ni⁺ columns or the maltose bindingprotein of the commercially available vector pMAL (New England BioLabs,Beverly, Mass.). A currently preferred, but by no means necessary,fusion domain is a filamentous phage membrane anchor. This domain isparticularly useful for screening phage display libraries of monoclonalantibodies but may be of less utility for the mass production ofantibodies. The filamentous phage membrane anchor is preferably a domainof the cpIII or cpVIII coat protein capable of associating with thematrix of a filamentous phage particle, thereby incorporating the fusionpolypeptide onto the phage surface, to enable solid phase binding tospecific antigens or epitopes and thereby allow enrichment and selectionof the specific antibodies or fragments encoded by the phagemid vector.

The secretion signal is a leader peptide domain of a protein thattargets the protein to the membrane of the host cell, such as theperiplasmic membrane of Gram-negative bacteria. The leader sequence ofthe pelB protein has previously been used as a secretion signal forfusion proteins (Better, M. et al. 1988 Science 240:1041-1043; Sastry,L. et al. 1989 Proc, Natl. Acad. Sci. (USA) 86:5728-5732; and Mullinax,R. L. et al., 1990 Proc. Natl. Acad. Sci. (USA) 87:8095-8099). Aminoacid residue sequences for other secretion signal polypeptide domainsfrom E. coli useful in this invention can be found in Neidhard, F. C.(ed.), 1987 Escherichia coli and Salmonella Typhimurium: TyphimuriumCellular and Molecular Biology, American Society for Microbiology,Washington, D.C.

To achieve high levels of gene expression in E. coli, it is necessary touse not only strong promoters to generate large quantities of mRNA, butalso ribosome binding sites to ensure that the mRNA is efficientlytranslated. In E. coli, the ribosome binding site includes an initiationcodon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotidesupstream from the initiation codon (Shine et al. 1975 Nature 254:34-38).The sequence, which is called the Shine-Dalgarno (SD) sequence, iscomplementary to the 3′ end of E. coli 16S rRNA. Binding of the ribosometo mRNA and the sequence at the 3′ end of the mRNA can be affected byseveral factors: the degree of complementarity between the SD sequenceand 3′ end of the 16S rRNA; the spacing lying between the SD sequenceand the AUG; and the nucleotide sequence following the AUG, whichaffects ribosome binding. The 3′ regulatory sequences define at leastone termination (stop) codon in frame with and operably joined to theheterologous fusion polypeptide.

In preferred embodiments with a prokaryotic expression host, the vectorutilized includes a prokaryotic origin of replication or replicon, i.e.,a DNA sequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extrachromosomally in aprokaryotic host cell, such as a bacterial host cell, transformedtherewith. Such origins of replication are well known in the art.Preferred origins of replication are those that are efficient in thehost organism. A preferred host cell is E. coli. For use of a vector inE. coli, a preferred origin of replication is ColEI found in pBR322 anda variety of other common plasmids. Also preferred is the p15A origin ofreplication found on pACYC and its derivatives. The ColEI and p15Areplicons have been extensively utilized in molecular biology and areavailable on a variety of plasmids and are described by Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory Press.

In addition, those embodiments that include a prokaryotic repliconpreferably also include a gene whose expression confers a selectiveadvantage, such as drug resistance, to a bacterial host transformedtherewith. Typical bacterial drug resistance genes are those that conferresistance to ampicillin, tetracycline, neomycin/kanamycin orchloramphenicol. Vectors typically also contain convenient restrictionsites for insertion of translatable DNA sequences. Exemplary vectors arethe plasmids pUC18 and pUC19 and derived vectors such as thosecommercially available from suppliers such as Invitrogen, (San Diego,Calif.).

When the antibodies of the invention include both heavy chain and lightchain sequences, these sequences may be encoded on separate vectors or,more conveniently, may be expressed by a single vector. The heavy andlight chain may, after translation or after secretion, form theheterodimeric structure of natural antibody molecules. Such aheterodimeric antibody may or may not be stabilized by disulfide bondsbetween the heavy and light chains.

A vector for expression of heterodimeric antibodies, such as thefull-length antibodies of the invention or the F(ab′)₂, Fab or Fvfragment antibodies of the invention, is a recombinant DNA moleculeadapted for receiving and expressing translatable first and second DNAsequences. That is, a DNA expression vector for expressing aheterodimeric antibody provides a system for independently cloning(inserting) the two translatable DNA sequences into two separatecassettes present in the vector, to form two separate cistrons forexpressing the first and second polypeptides of a heterodimericantibody. The DNA expression vector for expressing two cistrons isreferred to as a di-cistronic expression vector.

Preferably, the vector comprises a first cassette that includes upstreamand downstream DNA regulatory sequences operably joined via a sequenceof nucleotides adapted for directional ligation to an insert DNA. Theupstream translatable sequence preferably encodes the secretion signalas described above. The cassette includes DNA regulatory sequences forexpressing the first antibody polypeptide that is produced when aninsert translatable DNA sequence (insert DNA) is directionally insertedinto the cassette via the sequence of nucleotides adapted fordirectional ligation.

The dicistronic expression vector also contains a second cassette forexpressing the second antibody polypeptide. The second cassette includesa second translatable DNA sequence that preferably encodes a secretionsignal, as described above, operably joined at its 3′ terminus via asequence of nucleotides adapted for directional ligation to a downstreamDNA sequence of the vector that typically defines at least one stopcodon in the reading frame of the cassette. The second translatable DNAsequence is operably joined at its 5′ terminus to DNA regulatorysequences forming the 5′ elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion polypeptide comprising a secretion signal with apolypeptide coded by the insert DNA.

The antibodies of the present invention may additionally, of course, beproduced by eukaryotic cells such as CHO cells, human or mousehybridomas, immortalized B-lymphoblastoid cells, and the like. In thiscase, a vector is constructed in which eukaryotic regulatory sequencesare operably joined to the nucleotide sequences encoding the antibodypolypeptide or polypeptides. The design and selection of an appropriateeukaryotic vector is within the ability and discretion of one ofordinary skill in the art. The subsequent purification of the antibodiesmay be accomplished by any of a variety of standard means known in theart.

The antibodies of the present invention may furthermore, of course, beproduced in plants. In 1989, Hiatt et al. 1989, Nature 342:76-78 firstdemonstrated that functional antibodies could be produced in transgenicplants. Since then, a considerable amount of effort has been invested indeveloping plants for antibody (or “plantibody”) production (for reviewssee Giddings, G. et al., 2000 Nat Biotechnol 18:1151-1155; Fischer, R.and Emans, N., 2000, Transgenic Res 9:279-299). Recombinant antibodiescan be targeted to seeds, tubers, or fruits, making administration ofantibodies in such plant tissues advantageous for immunization programsin developing countries and worldwide.

In another embodiment, the present invention provides host cells, bothprokaryotic and eukaryotic, transformed or transfected with, andtherefore including, the vectors of the present invention.

Diagnostic and Pharmaceutical Anti-B5 or Anti-A33 Monoclonal AntibodyPreparations

The invention also relates to a method for preparing diagnostic orpharmaceutical compositions comprising the monoclonal antibodies of theinvention or polynucleotide sequences encoding the antibodies of theinvention or part thereof, the pharmaceutical compositions being usedfor immunoprophylaxis or immunotherapy of Orthopoxvirus infection. Thepharmaceutical preparation includes a pharmaceutically acceptablecarrier. Such carriers, as used herein, means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe active ingredients. The term “physiologically acceptable” refers toa non-toxic material that is compatible with a biological system such asa cell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

A preferred embodiment of the invention relates to monoclonal antibodieswhose heavy chains comprise in CDR3 the polypeptide having SEQ ID NO: 7,and/or whose light chains comprise in CDR3 the polypeptide having SEQ IDNO: 15; whose heavy chains comprise in CDR3 the polypeptide having SEQID NO: 23, and/or whose light chains comprise in CDR3 the polypeptidehaving SEQ ID NO: 31; whose heavy chains comprise in CDR3 thepolypeptide having SEQ ID NO: 39, and/or whose light chains comprise inCDR3 the polypeptide having SEQ ID NO: 47; whose heavy chains comprisein CDR3 the polypeptide having SEQ ID NO: 55, and/or whose light chainscomprise in CDR3 the polypeptide having SEQ ID NO: 63; whose heavychains comprise in CDR3 the polypeptide having SEQ ID NO: 71, and/orwhose light chains comprise in CDR3 the polypeptide having SEQ ID NO:79; and conservative variations of these peptides. The term“conservative variation” as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another orthe substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid provided that antibodies having thesubstituted polypeptide also bind or neutralize an Orthopoxvirus.Analogously, another preferred embodiment of the invention relates topolynucleotides which encode the above noted heavy chain polypeptidesand to polynucleotide sequences which are complementary to thesepolynucleotide sequences. Complementary polynucleotide sequences includethose sequences that hybridize to the polynucleotide sequences of theinvention under stringent hybridization conditions.

The 8AH8AL, 8AH7AL, 6C, 12C, or 12F antibody, or other anti-B5 oranti-A33 antibody of the invention may be labeled by a variety of meansfor use in diagnostic and/or pharmaceutical applications. There are manydifferent labels and methods of labeling known to those of ordinaryskill in the art. Examples of the types of labels which can be used inthe present invention include enzymes, radioisotopes, fluorescentcompounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically altered by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific antihapten antibodies.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise amonoclonal antibody of the invention that is, or can be, detectablylabeled. The kit may also have containers containing buffer(s) and/or acontainer comprising a reporter-means, such as a biotin-binding protein,such as avidin or streptavidin, bound to a reporter molecule, such as anenzymatic or fluorescent label.

In vitro Detection and Diagnostics

The monoclonal antibodies of the invention are suited for in vitro use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize the monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of Orthopoxvirus.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylase, natural andmodified cellulose, polyacrylamide, agarose and magnetite. The nature ofthe carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding monoclonal antibodies, or will be able to ascertain such,using routine experimentation.

For purposes of the invention, Orthopoxvirus may be detected by themonoclonal antibodies of the invention when present in biological fluidsand tissues. Any sample containing a detectable amount of Orthopoxviruscan be used. A sample can be a liquid such as urine, saliva,cerebrospinal fluid, blood, serum or the like; a solid or semi-solidsuch as tissues, feces, or the like; or, alternatively, a solid tissuesuch as those commonly used in histological diagnosis.

In vivo Detection of Orthopoxvirus

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the B5 or A33 antigen for which themonoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to Orthopoxvirusis detectable compared to the background. Further, it is desirable thatthe detectably labeled monoclonal antibody be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of infection of the individual. The dosage of monoclonal antibodycan vary from about 0.01 mg/kg to about 50 mg/kg, preferably 0.1 mg/kgto about 20 mg/kg, most preferably about 0.1 mg/kg to about 5 mg/kg.Such dosages may vary, for example, depending on whether multipleinjections are given, on the tissue being assayed, and other factorsknown to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting an appropriate radioisotope.The radioisotope chosen must have a type of decay which is detectablefor the given type of instrument. Still another important factor inselecting a radioisotope for in vivo diagnosis is that the half-life ofthe radioisotope be long enough such that it is still detectable at thetime of maximum uptake by the target, but short enough such thatdeleterious radiation with respect to the host is acceptable. Ideally, aradioisotope used for in vivo imaging will lack a particle emission butproduce a large number of photons in the 140-250 keV range, which may bereadily detected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions are the bifunctionalchelating agents such as diethylenetriaminepentacetic acid (DTPA) andethylenediaminetetra-acetic acid (EDTA) and similar molecules. Typicalexamples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr and²⁰¹Tl.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Crand ⁵⁶Fe.

The monoclonal antibodies of the invention can be used in vitro and invivo to monitor the course of Orthopoxvirus infection therapy. Thus, forexample, by measuring the increase or decrease in the number of cellsinfected with Orthopoxvirus or changes in the concentration ofOrthopoxvirus present in the body or in various body fluids, it would bepossible to determine whether a particular therapeutic regimen aimed atameliorating Orthopoxvirus infection is effective.

Prophylaxis and Therapy of Orthopoxvirus Infection

The monoclonal antibodies can also be used in prophylaxis and as therapyfor Orthopoxvirus infection in humans or other animals The terms,“prophylaxis” and “therapy” as used herein in conjunction with themonoclonal antibodies of the invention denote both prophylactic as wellas therapeutic administration and both passive immunization withsubstantially purified polypeptide products, as well as gene therapy bytransfer of polynucleotide sequences encoding the product or partthereof. Thus, the monoclonal antibodies can be administered tohigh-risk subjects in order to lessen the likelihood and/or severity ofOrthopoxvirus infection or administered to subjects already evidencingactive Orthopoxvirus infection. In the present invention, Fab fragmentsalso bind or neutralize Orthopoxviruses and therefore may be used totreat Orthopoxvirus infection but full-length antibody molecules areotherwise preferred.

As used herein, a “prophylactically effective amount” of the monoclonalantibodies of the invention is a dosage large enough to produce thedesired effect in the protection of individuals against Orthopoxvirusinfection for a reasonable period of time, such as one to two months orlonger following administration. A prophylactically effective amount isnot, however, a dosage so large as to cause adverse side effects, suchas hyperviscosity syndromes, pulmonary edema, congestive heart failure,and the like. Generally, a prophylactically effective amount may varywith the subject's age, condition, and sex, as well as the extent of theinfection in the subject and can be determined by one of skill in theart. The dosage of the prophylactically effective amount may be adjustedby the individual physician or veterinarian in the event of anycomplication. A prophylactically effective amount may vary from about0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about20 mg/kg, most preferably from about 0.2 mg/kg to about 5 mg/kg, in oneor more administrations (priming and boosting).

As used herein, a “therapeutically effective amount” of the monoclonalantibodies of the invention is a dosage large enough to produce thedesired effect in which the symptoms of Orthopoxvirus infection areameliorated or the likelihood of infection is decreased. Atherapeutically effective amount is not, however, a dosage so large asto cause adverse side effects, such as hyperviscosity syndromes,pulmonary edema, congestive heart failure, and the like. Generally, atherapeutically effective amount may vary with the subject's age,condition, and sex, as well as the extent of the infection in thesubject and can be determined by one of skill in the art. The dosage ofthe therapeutically effective amount may be adjusted by the individualphysician or veterinarian in the event of any complication. Atherapeutically effective amount may vary from about 0.01 mg/kg to about50 mg/kg, preferably from about 0.1 mg/kg to about 20 mg/kg, mostpreferably from about 0.2 mg/kg to about 5 mg/kg, in one or more doseadministrations daily, for one or several days. Preferred isadministration of the antibody for 2 to 5 or more consecutive days inorder to avoid “rebound” of virus replication from occurring.

The monoclonal antibodies of the invention can be administered byinjection or by gradual infusion over time. The administration of themonoclonal antibodies of the invention may, for example, be intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. Techniques for preparing injectate or infusate deliverysystems containing antibodies are well known to those of skill in theart. Generally, such systems should utilize components which will notsignificantly impair the biological properties of the antibodies, suchas the paratope binding capacity (see, for example, Remington'sPharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody injectates or infusates without resortto undue experimentation.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and thelike.

Chimpanzee Monoclonal Antibodies to Vaccinia Virus B5 Protein ProtectMice Against Vaccinia Virus and Neutralize Vaccinia and Smallpox Viruses

Chimpanzee Fabs against the B5 envelope glycoprotein of vaccinia viruswere isolated and converted into complete monoclonal antibodies (MAbs)with human γ1 heavy chain constant regions. The two MAbs (8AH8AL and8AH7AL) displayed high binding affinities to B5 (K_(d) of 0.2 nM and 0.7nM). The MAb 8AH8AL inhibited the spread of vaccinia virus as well asvariola virus (the causative agent of smallpox) in vitro, protected micefrom subsequent intranasal challenge with virulent vaccinia virus,protected mice when administered 2 days post-challenge and providedsignificantly greater protection than that afforded by a previouslyisolated rat anti-B5 MAb (19C2) or by vaccinia immune globulin. The MAbbound to a conformational epitope between amino acids 20 and 130 of B5.These chimpanzee/human anti-B5 MAbs are envisioned as providing thebasis for the prevention and treatment of vaccinia virus-inducedcomplications of vaccination against smallpox and in theimmunoprophylaxis and immunotherapy of smallpox.

Isolation and Characterization of Vaccinia B5-Specific Fabs

The chimpanzee Fab-displaying phage library was panned againstrecombinant VACV B5 protein (275t) and 96 individual clones wererandomly picked and screened for binding to B5 by phage ELISA with BSAas a negative control. Ninety percent of the clones preferentially boundto B5. DNA sequencing of the variable regions of heavy (VH) and light(VL) chains from 18 positive clones showed that a single VH gene waspaired with two different VL genes. These two clones were designated8AH8AL and 8AH7AL (GenBank accession numbers: DQ316791, DQ316789,DQ316792, and DQ316790). The sequences of VH and VL genes are shown inFIGS. 1 a and 1 b. A search in V-Base (Cook, G. P. & Tomlinson, I. M.1995 Immunol Today 16:237-242) indicated that the VH gene putativelyoriginated from germline gene V3-49, which belongs to the VH3 family;the two VL genes were from Vλ germline gene 2a2.272A12, which belongs tothe VX 2 family.

The Fab sequences were converted into full-length IgG as described inExample 1 and the IgGs were examined for their binding specificity byELISA. Anti-B5 8AH8AL bound to B5 protein with high specificity andaffinity, but not to unrelated proteins (BSA, thyroglobulin,phosphorylase b, lysozyme and cytochrome-c) (FIG. 1 c). The two anti-B5MAbs had the identical binding specificity.

Epitope Recognized by the Anti-B5 Mab

In the absence of differences in heavy chain sequence, 8AH8AL was chosenfor epitope mapping as it had a slightly higher affinity. Westernblotting was used to locate the epitope recognized by anti-B5 8AH8AL.Different B5 fragments generated by N- and C-terminal deletions wereproduced in bacteria. Western blotting with anti-His confirmed thatsimilar amounts of each peptide were tested for reaction with anti-B58AH8AL. As seen in FIG. 2 a and summarized in FIG. 2 b, the shortestpeptide that strongly reacted with anti-B5 8AH8AL consisted of residues20 to 130 on B5 protein. Therefore, it required 110 amino acid residuesto form the epitope, which suggested the epitope was conformationalsince a linear epitope is usually composed of 5-15 amino acid residues.

To address whether MAbs can react with its smallpox B5 counterpart, theB520-130 protein of VACV was converted to that of variola virus viasplicing by overlap extension PCR (Example 5). Vaccinia and variolavirus B520-130 proteins were quantified by Western blotting usingHRP-conjugated anti-His. The Western blotting with 8AH8AL showed thatanti-B5 MAb cross-reacted with smallpox B520-130, although the reactionwas not as strong as with VACV B520-130.

Binding Affinity and In Vitro Neutralizing Activity

The affinity of the two chimpanzee/human MAbs and a rat MAb (19C2) forbinding to VACV B5 protein was measured by surface plasmon resonance(SPR) biosensor. A K_(d) of 0.6 nM and a dissociation rate constant of˜10⁻⁵/sec was observed for 8AH8AL (Table 1). A similar K_(d) wasdetermined in the SPR solution competition assay, both for 8AH8AL (0.2nM) and for 8AH7AL (0.7 nM). In contrast, the affinity of the rat MAb19C2 was ˜13-fold weaker (Table 1). Remarkably, the off-rate of the MAb8AH8AL was 25-fold slower than that of the rat MAb 19C2. Thus, the halflife of the antibody-antigen complex was ˜19 h for the chimpanzee/humanMAbs and less than 45 min for the rat MAb.

TABLE 1 Antibody affinity of anti-B5 Mabs¹ Antibody k_(on) (M⁻¹s⁻¹)k_(off) (s⁻¹) K_(d) (nM) 8AH8AL 2 × 10⁴   1 × 10⁻⁵ 0.6 19C2 3 × 10⁴ 2.6× 10⁻⁴ 7.5 ¹Anti-B5 IgG of chimpanzee/human MAb 8AH8AL and a rat MAb19C2 were immobilized individually on the surface plasmon resonancesensor surfaces. The antibody binding responses to B5 (275t) proteinwere collected at a range of concentrations between 0.05 and 500 nM ofantigen. The kinetic rate constant of association (k_(on)) anddissociation (k_(off)) rates were measured from surface bindingkinetics, and the equilibrium dissociation constant (K_(d)) wascalculated as the ratio k_(off)/k_(on).

Since B5 is an EV-specific protein, in vitro neutralization activity ofanti-B5 MAbs was measured by the comet-reduction assay, an establishedmethod that measures the inhibition of comet-like plaque formation bythe released EV form of the virus (Appleyard, G. et al. 1971 J Gen Virol13:9-17; Law, M. et al. 2002 J Gen Virol 83:209-222). The EV of the IHDstrain of VACV formed comet-shaped plaques in the absence of antibodies,but the formation of comets was completely blocked by the addition of anexcess of rabbit hyperimmune serum to VACV (FIG. 3 a). The monoclonalanti-B5 clones, 8AH8AL and 8AH7AL, reduced the formation of comet-likeplaques of vaccinia virus EV at the lowest dose tested (FIG. 3 a).Similarly, the formation of comet-shaped plaques of the Solaimen strainof variola EV was inhibited by 8AH8AL in a dose-dependent manner (FIG. 3b), indicating that the anti-B5 MAbs possessed neutralizing activityagainst EV of both viruses.

Protection of Mice Against Challenge with Virulent VACV

The BALB/c mouse pneumonia model with VACV WR challenge (Smee, D. F. etal. 2001 Antiviral Res 52:55-62; Williamson, J. D. et al. 1990 J GenVirol 71:2761-2767) was used for the following reasons: weight loss anddeath are correlated with replication in the lungs, allowing the onsetand progress of disease to be monitored by a non-invasive method thatreduces the number of animals needed for significance (Law, M. et al.2005 J Gen Virol 86:991-1000); the model has been used for activeimmunization studies with live VACV as well as with individual VACVproteins (Fogg, C. et al. 2004 J Virol 78:10230-10237) and for passiveimmunization studies with antisera prepared against VACV and VACVproteins (Law, M. et al. 2005 J Gen Virol 86:991-1000), and theintranasal (IN) route is believed to be the major avenue fortransmission of variola virus. The two anti-B5 chimpanzee/human MAbs,8AH8AL, 8AH7AL, and a rat anti-B5 MAb, 19C2 (Schmelz, M. et al. 1994 JVirol 68:130-147) were compared for their in vivo protective activity.The control mice lost weight continuously starting at day 5 followingchallenge with 10⁵ pfu of WR and 2 of the 5 mice were sacrificed becausethey reached 70% of starting weight (FIG. 4 a). In contrast, the micethat were injected with MAbs 8AH8AL or 8AH7AL did not lose weight afterthe identical challenge with 10⁵ pfu of WR, indicating that fullprotection was achieved. Although the rat MAb 19C2 also protected micecompared to the control mice, substantial weight loss was observed. Thetwo chimpanzee/human MAbs provided significantly better protection thanthat provided by the rat MAb (P<0.0001 on day 8). The difference inweight loss between the no antibody control group and each of theimmunized groups was also highly significant on day 8 (P<0.0001).

Since there was no difference in protective efficiency between 8AH8ALand 8AH7AL, only the 8AH8AL MAb was used in determining the minimumeffective dose of anti-B5. The half-life of the MAb was found to be 6.4days in mice. Groups of mice were given decreasing doses of 8AH8AL (90,45, 22.5 μg per mouse) and a single 5 mg dose of human VIG (2.5× therecommended human dose on a weight basis) was used for comparison. All 5control mice died or were sacrificed when their weight fell to 70% ofstarting weight (FIG. 4 b). In contrast, all of the mice injected with8AH8AL, even at the lowest dose, or with VIG were protected from deathfollowing WR challenge. Protection against disease, as measured by thedegree of weight loss, however, was dose-dependent for 8AH8AL. Thedifference in weight loss between mice immunized with 8AH8AL andunimmunized control mice was highly significant on day 7 (P<0.0001 for90 and 45 μg, P=0.0005 for 22.5 μg). Five mg of VIG reduced weight lossafter challenge (P=0.003 on day 7). The difference in weight lossbetween mice receiving 5 mg of VIG and those receiving 45 μg or 90 μg of8AH8AL was highly significant on day 8 (P<0.0001). No statisticallysignificant difference was found between 5 mg of VIG and 22.5 μg of8AH8AL.

The therapeutic value of 8AH8AL was assessed by administration of theMAb two days after challenge with VACV (FIG. 4 c). A single 90 μg doseof 8AH8AL administered 48 h after infection protected the mice (P<0.0001on day 7, versus unimmunized controls) and they experienced only slightweight loss, followed by rapid recovery. In contrast, a single 5 mg doseof VIG administered 48 h after infection afforded much less protection(P=0.057 on day 7, versus unimmunized controls) and 2 of the 5 mice weresacrificed because their weight loss reached 30%. The difference inweight loss between the mice receiving the MAb and those receiving VIGwas highly significant on day 7 (P<0.0001), indicating that the MAb wasmore therapeutic than the VIG.

Convalescent sera collected 22 days after challenge from the micedescribed above (FIG. 4 b and c) contained negligible amounts of theinjected MAbs and were assayed for induced murine antibodies to twoEV-associated proteins, B5 and A33, and two MV-associated proteins, L1and A27, by ELISA The sera were also assayed for neutralizing antibodiesto the MV form of VACV. Only challenged mice that had receivedantibodies (MAb or VIG) were included since all of the non-immunizedchallenged mice had been sacrificed because of weight loss. Sera frommice not challenged with VACV served as negative controls.

Mice that had received VIG either before or after challenge mounted asignificant antibody response against all of the proteins tested (FIG. 5a-d), indicating that viral replication and production of VACV hadoccurred. In contrast, mice that had received the chimpanzee-derived MAbdid not mount a significant immune response to either of the two EVmembrane proteins, however, they did demonstrate an immune response toboth MV membrane-associated proteins, which varied according to the doseand time of MAb administered, suggesting that higher doses of the MAbinhibited virus replication better than lower doses. This dose-dependentpattern of response by the challenged mice was reflected also in theirneutralizing antibody response to MV (FIG. 5 e).

Discussion

Several studies have suggested that antibodies are sufficient to protectagainst orthopoxvirus infections in mice and monkeys (Lustig, S. et al.2005 J Virol 79:13454-13462; Wyatt, L. S. et al. 2004 Proc Natl Acad SciUSA 101:4590-4595; Edghill-Smith, Y. et al. 2005 Nat Med 11:740-747).Here, we demonstrate that chimpanzee MAbs against VACV B5 protein (anEV-specific protein) alone are sufficient not only to protect mice fromlethal challenge with virulent VACV, but also to confer therapeuticprotection of mice when administered two days after infection. Theresult is consistent with the previous finding that neutralizingantibodies against EV play a critical role in protective immunity(Appleyard, G. & Andrews, C. 1974 J Gen Virol 23:197-200; Turner, G. S.& Squires, E. J. 1971 J Gen Virol 13:19-25).

Our anti-B5 MAbs exhibited much higher protective efficacy than did arat anti-B5 MAb or human VIG. Competition ELISA showed thatchimpanzee/human and rat MAbs did not compete with each other forbinding to B5, suggesting that they recognize different epitopes. Inaddition, the chimpanzee/human MAbs had higher binding affinity than therat MAb. Noteworthy is that the chimpanzee/human MAbs had a 25-foldslower off-rate than the rat MAb. The difference in binding sites andaffinities between the chimpanzee and rat MAbs may contribute to theirdifferent protective efficacies. The likely reason that human VIG isinferior to the chimpanzee/human MAbs in animal studies is that theconcentration of protective antibodies in VIG is low. Indeed, based onELISA, we found that 5 mg of VIG contained the equivalent of less than10 μg of MAb to B5.

The MAbs to B5 inhibited VACV spread in tissue culture cells and theireffect in vivo could have a related explanation. We measured mouseantibodies to two EV membrane proteins (B5 and A33) and to two MVmembrane-associated proteins (L1 and A27) as a measure of virusreplication. Animals passively immunized with VIG (5 mg) raisedantibodies to all 4 proteins, indicating significant virus replication,which was consistent with the considerable weight loss of these animals.In contrast, antibodies to the VACV proteins were much lower in animalsthat received the highest amount of MAb (90 μg) and exhibited minimalweight loss. More intriguing were the results obtained with animalsreceiving 22.5 or 45 μg of MAb. These animals also did not make aresponse to either of the EV membrane proteins, but did make adose-dependent response to the MV membrane-associated proteins. Thereare several possible explanations for this dichotomy. The simplest isthat EV membrane proteins are less immunogenic than MV proteins andhigher amounts of virus replication are needed for a response. However,the protection achieved with the low dose MAb and VIG was notstatistically different. An alternative explanation for the differencein antibody response to EV-specific proteins is that the B5 MAbaggregated progeny EV on the infected cell surface and prevented theinduction of antibodies to EV membrane proteins specifically. Indeed,agglutination of progeny EV on the surface of infected cells has beensuggested as the mechanism by which EV antibodies prevent the formationof comet plaques (Law, M. & Smith, G. L. 2001 Virology 280:132-142).

The use of anti-B5 MAbs in treatment of smallpox vaccine-associatedcomplications would overcome the limitations posed by VIG, such as a lowtiter of neutralizing activity, variability and risk of transmission ofinfectious agents. It is especially important that anti-B5 MAbscross-reacted with variola virus B5 and neutralized variola virus invitro. Amino acid sequence comparison of B5 at residues 20 to 130 (aneutralization epitope recognized by the anti-B5 MAb) from vaccinia,variola and monkeypox viruses revealed that there are 10 amino aciddifferences between vaccinia and variola viruses, but only 4 amino aciddifferences between vaccinia and monkeypox viruses, and 3 of these arethe same as in variola virus. Therefore, it is reasonable to assume thatanti-B5 MAb would neutralize monkeypox virus also since it canneutralize variola virus. It is conceivable that an anti-B5 MAb alone orin conjunction with other MAbs could be used directly in treatment ofbioterrorist-associated smallpox or in case of a monkeypox outbreak(Perkins, S. 2003 Contemp Top Lab Anim Sci 42:70-72).

Our anti-B5 MAb recognized a conformational epitope that is locatedbetween residues 20 and 130. Previously, two major neutralizing epitopesin B5 had been identified by testing a panel of 26 mouse anti-B5 MAbs;one epitope is localized to the SCR1-SCR2 border and the other islocated in the stalk region (Aldaz-Carroll et al. 2005 J Virol79:6260-6271). The neutralization epitope recognized by thechimpanzee/human MAb may be different from those previously reported(Aldaz-Carroll et al. 2005 J Virol 79:6260-6271). However, it is notpossible to make a direct comparison because of the different mappingmethods employed. Our method is based on differential binding of the MAbto a series of N- and C-terminally deleted peptides and the smallestpeptide that still reacted strongly with the MAb was considered to be abinding site whereas the other method is based on differential bindingof a MAb to a series of synthetic, linear overlapping peptides(Aldaz-Carroll et al. 2005 J Virol 79:6260-6271).

In summary, we have generated from the bone marrow of two immunizedchimpanzees human-like MAbs that neutralize the extracellular form ofVACV as well as that of variola virus. The MAbs protect mice from lethalchallenge with virulent VACV and are therapeutic when administered twodays after exposure. These MAbs provide the first alternative to VIG fortreatment of complications of smallpox vaccination and a basis for theprevention and treatment of smallpox.

Chimpanzee Monoclonal Antibodies to Vaccinia Virus A33 Protein ProtectMice Against Vaccinia Virus and Neutralize Vaccinia and Smallpox VirusesIsolation and Characterization of Vaccinia A33-Specific Fabs

The chimpanzee Fab-displaying phage library was panned againstrecombinant vaccinia virus (VACV) A33 protein and 96 individual cloneswere randomly picked and screened for binding to A33 by phage ELISA withBSA as a negative control. Ninety percent of the clones preferentiallybound to A33. DNA sequencing of the variable regions of heavy (VH) andlight (VL) chains from 16 positive clones showed that there were threedistinct clones. These three clones were designated 6C, 12C, and 12F.The sequences of VH and VL genes are shown in FIGS. 6 a and 6 b. Theclosest human germline gene for each VH and VL gene was identified bysearching V-Base database (Cook, G. P. & Tomlinson, I. M. 1995 ImmunolToday 16:237-242) (Table 2).

TABLE 2 Human Ig Germ Line Genes Most Closely Related to ChimpanzeeHeavy and Light Chains of Anti-A33 Mabs. VH VH D JH V_(λ) V_(λ) J_(λ)MAb Family Segment Segment Segment Family Segment Segment  6C VH7VI-4.1B D3-10 J5b V_(λ) I 1b.366F5 J_(λ) 3b 12C VH1 DP-25 D3-10 J4bV_(λ) III 3r.9C5 J_(λ) 2/3a 12F VH5 DP-73 D3-3  J5b V_(λ) II 2a2.272A12J_(λ) 2/3a The closest human VH and V_(λ) germ line genes wereidentified by V-BASE database.

The Fab sequences were converted into full-length IgG with human γ1constant regions and the IgGs were examined for their bindingspecificity by ELISA. Anti-A33 bound to A33 protein with highspecificity, but not to unrelated proteins (BSA, thyroglobulin,phosphorylase b, lysozyme and cytochrome-c) (FIG. 7). The other twoanti-A33 MAbs had the identical binding specificity.

Epitope Recognized by the Anti-A33 MAb

Competition ELISA indicated that the three MAbs may recognize the sameor closely related epitopes since they compete with each other forbinding to A33 protein. Therefore, 6C was chosen for epitope mapping asit has been used for neutralization assay extensively. His-taggedsoluble A33 peptides generated by N- and C-terminal deletions wereproduced in bacteria and affinity-purified through a nickel column.Western blotting with anti-His confirmed the identity of each peptide.However, the peptides did not react with anti-A33 MAb 6C in Westernblots, which suggests that the epitope recognized by the anti-A33 MAb isconformational. To date, the shortest peptide that reacted strongly withMAb 6C in ELISA consisted of amino acids 99-185 (FIG. 8).

Binding Affinity and In Vitro Neutralizing Activity

The affinity of the three chimpanzee/human MAbs for binding A33 proteinwas measured by surface plasmon resonance (SPR) biosensor. K_(d) rangeof 0.14 nM to 20 nM and a dissociation rate constant of ˜10⁻⁵/sec wasobserved for the three MAbs (Table 3).

TABLE 3 Binding affinities of anti-33 Mabs. MAb k_(on) (M⁻¹s⁻¹)k_(off)(s⁻¹) K_(d)  6C 6.8 × 10³ 1.35 × 10⁻⁴   20 nM 12C 4.6 × 10⁴ 2.15× 10⁻⁵ 0.46 nM 12F 1.85 × 10⁵  2.58 × 10⁻⁵ 0.14 nM

Since A33 is an EV-specific protein, in vitro neutralization activity ofanti-B5 MAbs was measured by the comet-reduction assay, an establishedmethod that measures the inhibition of comet-like plaque formation bythe released EV form of the virus (Appleyard, G. et al. 1971 J Gen Virol13:9-17; Law, M. et al. 2002 J Gen Virol 83:209-222). The EV of the IHDstrain of VACV formed comet-shaped plaques in the absence of antibodies,but the formation of comets was completely blocked by the addition of anexcess of rabbit hyperimmune serum to VACV (FIG. 9 a). The monoclonalanti-A33 clones, 6C, 12C and 12F, reduced the formation of comet-likeplaques of vaccinia virus EV in a dose-dependent manner (FIG. 9 a).Similarly, the formation of comet-shaped plaques of the Solaimen strainof variola EV was inhibited by 6C in a dose-dependent manner (FIG. 9 b),indicating that the anti-A33 MAbs possessed neutralizing activityagainst EV of both viruses.

Protection of mice against challenge with virulent VACV

The BALB/c mouse pneumonia model of VACV challenge (Smee, D. F. et al.2001 Antiviral Res 52:55-62; Williamson, J. D. et al. 1990 J Gen Virol71:2761-2767) was used for the following reasons: weight loss and deathare correlated with replication in the lungs, allowing the onset andprogress of disease to be monitored by a non-invasive method thatreduces the number of animals needed for significance (Law, M. et al.2005 J Gen Virol 86:991-1000); the model has been used for activeimmunization studies with live VACV as well as with individual VACVproteins (Fogg, C. et al. 2004 J Virol 78:10230-10237) and for passiveimmunization studies with antisera prepared against VACV and VACVproteins (Law, M. et al. 2005 J Gen Virol 86:991-1000), and theintranasal (IN) route is believed to be the major avenue fortransmission of variola virus. Groups of five BALB/c mice wereinoculated intraperitoneally with 90 μg of purified IgG ofchimpanzee/human MAbs 6C, 12C, 12F, or mouse MAb 1G10⁸ or 5 mg of humanvaccinia immune globulin (VIG). After 24 h, the mice were inoculatedintranasally with 10⁵ pfu of vaccinia virus, strain WR. Mice wereweighed individually and mean percentages of starting weight wereplotted. Controls were unimmunized (no antibody) or unchallenged (novirus). Mice that died naturally or were killed because of 30% weightloss are indicated (t). The control mice lost weight continuouslystarting on day 5 following challenge and 2 of the 5 mice weresacrificed because they reached 70% of starting weight (FIG. 10). Incontrast, the mice that were injected with MAbs 6C or 12F did not loseweight after the identical challenge, indicating that full protectionwas achieved. The mice receiving 12C experienced only slight weightloss, followed by rapid recovery. The mice receiving the mouse MAb 1G10or human VIG were protected, but the protective efficacy was less thanthat afforded by the chimpanzee/human MAbs.

Example 1 Reagents

Recombinant truncated B5 protein (275t) consisting of amino acids 20 to275 was produced in a baculovirus expression system (Aldaz-Carroll etal. 2005 J Virol 79:6260-6271) and was used as a panning antigen forselection of B5-reactive phage. Restriction and other enzymes were fromNew England BioLab (Beverly, Mass.). Oligonucleotides were synthesizedby Invitrogen (Carlsbad, Calif.). Anti-His horseradish peroxidase (HRP)conjugate, anti-human Fab HRP conjugate and anti-human Fab agarose werepurchased from Sigma (St. Louis, Mo.). VACV WR (ATCC VR-1354), IHD-J(from S. Dales, Rockefeller University), and VV-NP-siinfekl-EGFP weregrown in HeLaS3 cells (ATCC CCL-2.2), purified, and titered in BS-C-1cells as described (Earl, P. L. et al. 1998 Current Protocols inMolecular Biology (Greene & Wiley, New York)). A rat anti-B5 MAb, fromhybridoma 19C2 (Schmelz, M. et al. 1994 J Virol 68:130-147), waspurified from ascitic fluid (Taconic Biotechnology, Germantown, N.Y.).VIG (Cangene) was obtained from the Centers for Disease Control (CDC)(C. Allen, Drug Service, Atlanta, Ga.).

Animals

Chimpanzees 3863 and 3915 were immunized twice approximately 19 yearsapart (initially at Bioqual, Inc, Rockville, Md. and subsequently at theUniversity of Texas M.D. Anderson Cancer Center, Bastrop, Tex.) withVACV WR (Moss, B. et al. 1984 Nature 311:67-69). Bone marrow wasaspirated from the iliac crests of these animals 11 weeks after thesecond immunization. Mice were purchased from Taconic Biotechnology(Germantown, N.Y.). All animal experiments were performed underprotocols approved by the respective institutions as well as by theNIAID Animal Care and Use Committee.

Library Construction and Selection

Fab-encoding gene fragments were amplified from the cDNA of chimpanzeebone marrow-derived lymphocytes and cloned into pComb3H vector (Barbas,C. F. et al. 1991 Proc Natl Acad Sci USA 88:7978-7982; Schofield, D. J.et al. 2000 J Virol 74:5548-5555). The phage library was panned againstB5 protein and specific phage clones were selected as described(Harrison, J. L. et al. 1996 Methods Enzymol 267:83-109). The details oflibrary construction and selection are provided in Example 2.

Sequence Analysis

The genes encoding the variable region of the heavy (VH) and light (VL)chains of B5-specific clones were sequenced, and their correspondingamino acid sequences were aligned. The presumed family usage andgermline origin were established for each VH and VL gene by search ofV-Base (Cook, G. P. & Tomlinson, I. M. 1995 Immunol Today 16:237-242).

Expression and Purification of Fab and IgG

The phagemid containing λ light chain and γ1 heavy chain was cleavedwith NheI and SpeI and recircularized following removal of the phagegene III DNA fragment from the vector in order to encode soluble Fab.Bacteria containing circularized DNA without phage gene III werecultured in 2×YT medium containing 2% glucose, 100 μg/ml ampicillin and15 μg/ml tetracycline at 30° C. until the OD₆₀₀ reached 0.5-1. Theculture was diluted 5-fold in 2×YT medium without glucose and containing0.2 mM isopropyl β-D-thiogalactoside (IPTG) and culture was continued at27° C. for 20 h for expression of soluble Fab. Since the Fab was taggedat the C-terminus with (His)₆, the expressed proteins were readilyaffinity-purified on a nickel-charged column.

The conversion of Fab to full-length IgG was achieved by digestion of γ1Fd with XhoI and ApaI and cloning it into pCDHC68B vector (Trill, J. J.et al. 1995 Curr Opin Biotechnol 6:553-560), which contains the humanheavy chain constant region; the λ-chain was cloned into pCNHLCVector3(Trill, J. J. et al. 1995 Curr Opin Biotechnol 6:553-560) at XbaI andSad sites. For full-length IgG expression and purification, plasmidscontaining heavy chain and light chain were co-transfected into 293Tcells for transient expression. The IgG was purified by affinitychromatography with anti-human Fc agarose (Sigma).

The purity of the Fab and IgG was determined by SDS-PAGE and the proteinconcentration was determined by BCA assay (Pierce, Rockford, Ill.) andspectrophotometer measurement at OD₂₈₀.

ELISA Assay

B5 (275t) and non-related proteins (BSA, cytochrome-c, thyroglobulin,lysozyme, phosphorylase b) were coated in a 96-well plate by placing 100μl containing 1-5 μg/ml protein in 1×PBS, pH7.4 in each well andincubating the plate at room temperature (RT) overnight. Serialdilutions of soluble Fab, IgG or phage were added to the wells andplates were incubated for 2 h at RT. The plates were washed and thesecondary antibody conjugate (anti-His-HRP, anti-human Fab-HRP, oranti-M13-HRP) was added and incubated for 1 h at RT. The plates werewashed and the color was developed by adding TMB (Sigma). The plateswere read at OD450 in an ELISA plate reader.

Affinity Measurement

SPR biosensing experiments were conducted with a Biacore 3000 instrument(Biacore, Piscataway, N.J.) using short carboxy-methylated dextransensor surfaces (CM3, Biacore) and standard amine coupling as describedin detail elsewhere (Schuck, P. et al. 1999 in Current Protocols inProtein Science (John Wiley & Son, New York)). The procedure isdescribed in Example 3.

Epitope Mapping

The epitope recognized by anti-B5 8AH8AL was mapped by Western blot. B5peptides corresponding to aa 20-275, 20-160, 20-130, 20-100, 33-275,56-275, 71-275 were synthesized in E. coli as described previously(Zhou, Y. H. et al. 2004 Vaccine 22:2578-2585). The analysis isdescribed in Example 4.

Comet Reduction Assay for VACV

Monolayers of BS-C-1 cells in 6-well cell culture plates were infectedwith the IHD-J strain of VACV, which releases more EV than the WRstrain, at 50 to 100 plaque-forming units per well in Minimal EssentialMedium containing 2.5% FBS (MEM-2.5). After incubation for 2 h at 37°C., the medium was aspirated; cells were washed twice, and overlaid withMEM-2.5 containing the antibodies to be tested. The plates were thenplaced in a CO2 incubator for 36 h. Comets were visualized by stainingthe monolayers with a solution of 0.1% crystal violet in 20% ethanol.Each MAb was tested at several concentrations (5-30 pig per well).Rabbit polyclonal hyperimmune serum was used as a positive control.

Comet Reduction Assay for Variola Virus

The experiment was carried out in a BSL-4 smallpox laboratory at theCDC. Monolayers of BS-C-40 cells in 6-well cell culture plates wereinfected with the Solaimen strain of variola virus at 50 plaque-formingunits per well in RPMI medium containing 2% FBS. After 1 h, the mediumwas aspirated; cells were washed twice, and overlaid with RPMIcontaining antibody at different concentrations. Each treatment wasduplicated. The plates were then incubated at a fixed angle in a CO₂incubator for 4 days at 35.5° C. Cells were fixed and reacted withpolyclonal rabbit anti-variola antibody (Yang, H. et al. 2005 J ClinInvest 115:379-387). Following incubation with goat anti-rabbit-HRPconjugate, comets were visualized by addition of TruBlue peroxidasesubstrate (KPL).

Passive Immunization and Challenge with VACV Strain WR

Groups of seven-week old female BALB/c mice (Taconic Biotechnology,Germantown, N.Y.) were inoculated intraperitoneally with antibodydiluted in PBS. Non-immunized controls were injected with the samevolume of PBS. Either 24 h after or 48 h before immunization, mice werechallenged intranasally with 10⁵ plaque forming units (PFU) of VACV WRas described (Fogg, C. et al. 2004 J Virol 78:10230-10237). Mice wereweighed daily for 16 days and sacrificed if their weight diminished to70% of the initial weight, in accordance with NIAID Animal Care and Useprotocols. Mice were bled 24 h after passive immunization to monitoradministered antibody levels and on day 22 to measure development ofantibodies to the challenge virus.

Evaluation of Murine Convalescent Antibody Response Following Challenge

Serum samples taken from mice 22 days after challenge with VACV (seeabove) were analyzed for induction of mouse antibodies to recombinantproteins B5, A33, L1 (Aldaz-Carroll, L. et al. 2005 Virology 341:59-71),and A27 and neutralizing antibodies against MV. The recombinant proteinswere used to coat 96-well plates as described (Fogg, C. et al. 2004 JVirol 78:10230-10237) and two-fold serial dilutions of sera were addedto the plates. The bound mouse antibodies were detected by anti-mouseIgG(γ)-peroxidase (Roche, Indianapolis, Ind.). The substrate3,3′,5,5′-tetramethylbenzidine (BM Blue, POD, Roche) was used andendpoint titers were calculated as the dilution with absorbance (A₃₇₀and A₄₉₂) values two standard deviations above that measured in wellswithout antibodies. The IC₅₀ values for neutralization of the MV form ofVACV were determined by flow cytometry using the reporter virusVV-NP-siinfekl-EGFP as described (Earl, P. L. et al. 2003 J Virol77:10684-10688).

Statistical Analysis

Statistical differences in weight loss between groups of mice wereassessed by analysis of variance (ANOVA) using StatView software (SASInstitute, Inc., Cary, N.C.).

Example 2

Bone marrow-derived lymphocytes were isolated by Ficoll gradientseparation. Messenger RNA was extracted from 10⁸ lymphocytes with anmRNA purification kit (Amersham Biosciences, Piscataway, N.J.). The 1ststrand cDNA was synthesized from mRNA with a first-strand cDNA synthesiskit from Amersham Biosciences. The γ1 heavy chain Fd cDNA was amplifiedby polymerase chain reaction (PCR) using nine human γ1 heavychain-specific 5′ primers in combination with a chimpanzee γ1-specific3′ primer. The λ chain cDNA was amplified by PCR with seven human λchain-specific 5′ primers and a 3′ primer matching the end of theconstant region (Barbas, C. F. et al. 1991 Proc Natl Acad Sci USA88:7978-7982; Schofield, D. J. et al. 2000 J Virol 74:5548-5555). PCRwas performed for 30 cycles at 95° C., 1 min, 52° C., 1 min, and 72° C.,1 min with AmpliTaq DNA polymerase (Applied Biosystems, Foster City,Calif.). Chimpanzee 2 light chain and γ1 heavy chain DNA fragments werecloned into the pComb3H phagemid as described previously (Barbas, C. F.et al. 1991 Proc Natl Acad Sci USA 88:7978-7982; Schofield, D. J. et al.2000 J Virol 74:5548-5555). Briefly, amplified 2, chain DNA fragmentswere pooled, purified, digested with Sad and XbaI and cloned intopComb3H at the Sad and XbaI sites. The recombinant plasmid DNA wasintroduced into Esherichia coli Top 10 (Invitrogen) by electroporation,yielding 1×10⁷ individual clones. The plasmid DNA containing λ lightchain sequences was digested with XhoI and SpeI, and γ1 heavy chain DNAcut with the same enzymes was inserted. The plasmid DNA containing bothlight and heavy chains was transformed into E. coli Top10 byelectroporation, resulting in a Fab-displaying library with 5×10⁷individual clones.

The phagemid was rescued by superinfection with helper phage, VCS-M13(Stratagene, La Jolla, Calif.) and phagemids carrying Fab on their tipswere subjected to panning on B5 (275t) protein coated on ELISA wells.Nonspecifically adsorbed phages were removed by extensive washing.Specifically bound phages were eluted with 100 mM triethylamine,neutralized to pH 7.5, amplified and used for further selection asdescribed (Harrison, J. L. et al. 1996 Methods Enzymol 267:83-109).After three rounds of panning, randomly picked single Fab-bearing phageclones were screened for specific binding to B5 (275t) by ELISA.Briefly, 96-well ELISA plates were coated with 0.1 μg of B5 (275t) orcontrol protein BSA and blocked with PBS containing 3% nonfat milk.Fab-bearing phages in PBS containing 3% nonfat milk were added.Specifically bound phages were detected by adding HRP-conjugated mouseanti-M13, and the color was developed by adding tetramethylbenzidine(TMB) substrate. Absorbance at 450 nm was measured after addingsulphuric acid. Clones that differentially bound to B5 with A450 valuesof >1.0 were scored as positive, whereas values of <0.2 were scored asnegative.

Example 3 Affinity Measurement

Surfaces were activated with N-hydroxysuccinimide andN-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride, reactedwith antibody solutions at low ionic strength at pH 5.0, and unreactedgroups were quenched with ethanolamine. Binding experiments wereconducted at a flow rate of 5 μl/min in 10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.005% v/v surfactant P20 at pH 7.4 and 25° C., and the surfacewas regenerated with 10 mM glycine-HCl pH 2.0.

Anti-B5 IgG was immobilized to the surface and the kinetics of bindingand dissociation of B5 (275t) was recorded for 50 min and 3 h,respectively, at B5 (275t) concentrations between 0.05 and 500 nM. Inorder to eliminate the effect of immobilization-induced surface siteheterogeneity in the determination of the binding constants (Schuck, P.1997 Annu Rev Biophys Biomol Struct 26:541-566), the kinetic traces wereglobally fitted with a model for continuous ligand distributions(Svitel, J. et al. 2003 Biophys J 84:4062-4077). This resulted inresiduals of the global fit that were evenly distributed and of amagnitude in the order of noise of the data acquisition(root-mean-square deviation below 0.5 RU). No terms for mass transportlimitation were necessary. Since the 8AH8AL exhibited very slowkinetics, long extrapolation is required to determine the equilibriumconstants from the kinetic assay. Therefore, as a control, a solutioncompetition experiment was conducted, where soluble IgG at differentconcentrations was pre-incubated with B5 for 24 h, and the concentrationof unbound B5 was determined from the initial slope of the surfacebinding when passing the mixture over the anti-B5 functionalizedsurface. The standard competition isotherm model was used for theanalysis (Schuck, P. 1997 Annu Rev Biophys Biomol Struct 26:541-566;Schuck, P. et al. 1999 in Current Protocols in Protein Science (JohnWiley & Son, New York)).

Example 4 Epitope Mapping

The epitope recognized by anti-B5 8AH8AL was mapped by Western blot. B5peptides corresponding to aa 20-275, 20-160, 20-130, 20-100, 33-275,56-275, and 71-275 were synthesized in E. coli as described previously(Zhou, Y. H. et al. 2004 Vaccine 22:2578-2585). In brief, B5R DNAfragments encoding the above peptides were amplified from B5R cDNA(Isaacs, S, N. et al. 1992 J Virol 66:7217-7224) with primers containingPstI and HindIII sites at 5′- and 3′-ends, respectively, by PCR. PCRproducts were inserted into pRESET vector (Invitrogen) at PstI andHindIII sites. The sequence encoding a six-histidine tag in the vectorwas in frame with the insert DNA for easy detection and purification.The recombinant plasmid DNA carrying the B5R DNA insert was transformedinto E. coli JM109 and the sequence of the insert was confirmed.

The recombinant plasmid DNA was subsequently transformed into E. coliBL21(DE3)pLysS (Invitrogen) for expression. In brief, the bacteria werecultured at 37° C. in SOB medium containing ampicillin andchloramphenicol and the expression was induced by IPTG. The bacteriawere collected and resuspended in SDS-PAGE sample buffer. The amount ofB5 peptides was estimated by SDS-PAGE (16% Tris-Glycine gel, Invitrogen)and Western blotting with HRP-conjugated anti-His. Approximately equalamounts of protein were separated by SDS-PAGE, transferred tonitrocellulose membrane, and probed with anti-B5 8AH8AL. The bound MAbon the membrane was detected by HRP-conjugated anti-human IgG F(ab′)2(Sigma). Following reaction with LumiGLO chemiluminescent peroxidasesubstrate (KPL, Gaithersburg, Md.), the positive bands were detected byexposing the membrane to X-ray film.

Example 5

Truncated Smallpox B5 Constructed from Vaccinia Counterpart

To construct a truncated B5 protein with the smallpox virus sequence,eight primers containing all of the 10 amino acids that are specific forsmallpox B5 were designed as follows:

(SEQ ID NO: 81) B5-20F 5′-AACTGCAGACATGTACTGTACCCACTATG-3′(SEQ ID NO: 82) MT-1F 5′-TGATTCGGGATATTATTCTTTGGATCC-3′ (SEQ ID NO: 83)MT-2R 5′-GGATCCAAAGAATAATATCCCGAATCA-3′ (SEQ ID NO: 84) MT-3F5′-ACAGTTTCTGATTACGTCTCTGAA-3′ (SEQ ID NO: 85) MT-4R5′-TTCAGAGACGTAATCAGAAACTGT-3′ (SEQ ID NO: 86) MT-5F5′-AATGCCATCATCACACTAATTTGCAAGGACGAA-3′ (SEQ ID NO: 87) MT-6R5′-TTCGTCCTTGCAAATTAGTGTGATGATGGCATT-3′ (SEQ ID NO: 88) B5-130R5′-AAAAGCTTACATTCCGCATTAGGACACGT-3′

Thirty cycles of PCR (94° C. for 25 seconds, 50° C. for 15 seconds, and72° C. for 20 seconds) with primer pairs of B5-20F/MT-2R, MT-1F/MT-4R,MT-3F/MT-6R, and MT-5F/B5-130R generated 4 PCR products. The DNAfragment encoding amino acid residues 20-130 of smallpox B5 was createdby overlapping extension PCR of the 4 PCR products. Briefly, equalamounts of the gel-purified PCR products were mixed with two flankingprimers, B5-20F and B5-130R, and the mixture was amplified for 30 cycles(94° C. for 25 seconds, 55° C. for 15 seconds, and 72° C. for 1 min) Thefull-length PCR product was purified and cloned into pRESET vector andthe DNA sequence of the insert was confirmed. The methods for proteinexpression and Western blotting analysis have been described in theepitope mapping section.

Example 6

To determine the minimum effective dose of anti-A33 6C, groups of mice(5 mice per group) were given decreasing amounts of 6C (90, 45, 22.5 μgper mouse) or a single 5 mg dose of human VIG (2.5× the recommendedhuman dose on a weight basis). Twenty four hours later, the animals werechallenged intranasally with 10⁵ PFU of VACA WR. Mice were weighed dailyand the weight loss was used to estimate the protective efficacy. Asshown in FIG. 11, mice receiving either mAb 6C or VIG had statisticallysignificant less weight loss than the unimmunized mice (P<0.0001 at day8). Better protection was achieved by mAb 6C than by VIG since almost noweight loss was observed for the mice given mAb 6C even at the lowestamount tested (22.5 μg), whereas the mice given VIG lost substantialweight. The difference in weight loss between mice given mAb 6C at alldifferent doses and mice given VIG was statistically significant on day8 (P<0.0001).

To assess the therapeutic value of mAb 6C, the mAb or human VIG wasadministered to mice 2 days after challenge with VACA WR (FIG. 12). Micegiven 6C had much less weight loss than those given VIG and thedifference was statistically significant (P=0.002, on day 9).Nevertheless, both 6C and VIG provided significant protection asmeasured by weight loss (P<0.0001 on day 8,6C-treated or VIG-treatedversus untreated).

To determine if there was a synergistic effect between anti-B5 mAb8AH8AL and anti-A33 6C, we compared the protective efficacy of theindividual mAbs and VIG with that of a combination of the two mAbs inmice challenged with WR before and after administration of antibody(FIG. 13 and FIG. 14). The protective efficacy of two mAbs was notgreater than that of the individual mAbs. mAb 6C appeared to protectslightly better than mAb 8AH8AL (P=0.0189 on day 10) when administeredbefore challenge, whereas, 8AH8AL provided much stronger protection than6C when administered after challenge (P<0.0001 on day 8).

Example 7

Anti-A33 MAb 12F had about 140-fold higher affinity than MAb 6C (Table3). To determine if affinity was related to the protective efficacy ofthe antibodies, MAb 12F and MAb 6C were tested in parallel in the mousepneumonia model at different doses. As shown in FIG. 15, the protectionprovided by 12F was no different from that provided by 6C. The possiblereason for this result may be the dimeric nature of A33 protein, whichmakes antibody avidity, rather than affinity the dominant binding force.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

1. A substantially pure polypeptide comprising a fully human orhumanized chimpanzee monoclonal antibody that binds A33 antigen, whereinsaid monoclonal antibody comprises a heavy chain CDR1 region having theamino acid sequence of SEQ ID NO:67, a heavy chain CDR2 region havingthe amino acid sequence of SEQ ID NO:69, a heavy chain CDR3 regionhaving the amino acid sequence of SEQ ID NO:71, a light chain CDR1region having the amino acid sequence of SEQ ID NO:75, a light chainCDR2 region having the amino acid sequence of SEQ ID NO:77 and a lightchain CDR3 region having the amino acid sequence of SEQ ID NO:79. 2-3.(canceled)
 4. A substantially pure polypeptide comprising a monoclonalantibody that binds the antigen to which anti-vaccinia virus A33 12Fprotein Fab fragment deposited with the ATCC as ATCC Accession No.PTA-7324 binds.
 5. The substantially pure polypeptide of claim 1 whereinsaid antibody comprises a Fd fragment.
 6. The substantially purepolypeptide of claim 1 wherein said antibody comprises a Fab fragment.7-21. (canceled)
 22. An anti-vaccinia virus A33 12F deposited with ATCCas ATCC Accession No. PTA-7324.
 23. A pharmaceutical preparationcomprising the monoclonal antibody of claim
 1. 24. A diagnosticpreparation comprising the monoclonal antibody of claim
 1. 25. A methodfor inhibiting Orthopoxvirus infection comprising: administering to apatient an effective amount of the pharmaceutical preparation of claim23 to inhibit said Orthopoxvirus infection.
 26. (canceled)
 27. A methodfor the diagnosis of Orthopoxvirus infection comprising: obtaining thediagnostic preparation of claim 24; administering to a patient aneffective amount of the diagnostic preparation; and detecting binding ofthe monoclonal antibody as a determination of a presence ofOrthopoxvirus.
 28. A method of detecting the presence of Orthopoxvirusin a biological sample comprising: obtaining the diagnostic preparationof claim 24; contacting said sample with the diagnostic preparation; andassaying binding of the antibody as a determination of the presence ofsaid Orthopoxvirus.
 29. The substantially pure polypeptide of claim 1,wherein said monoclonal antibody comprises a V_(H) region having theamino acid sequence of SEQ ID NO:65.
 30. The substantially purepolypeptide of claim 1, wherein said monoclonal antibody comprises aV_(L) region having the amino acid sequence of SEQ ID NO:73.
 31. Asubstantially pure monoclonal antibody that binds a conformationalepitope of A33 antigen to which the monoclonal antibody of claim 1binds.